Male tissue-preferred regulatory region and method of using same

ABSTRACT

The present invention relates to an isolated nucleic acid sequence encoding the Ms45 male tissue-preferred regulatory region. In one aspect this invention relates the use of this male tissue-preferred regulatory region in mediating fertility. An example of such use is the production of hybrid seed such as in a male sterility system. The Ms45 male tissue-preferred regulatory region can be operably linked with exogenous genes, such as those encoding cytotoxins, complementary nucleotidic units and inhibitory molecules. This invention also relates to plant cells, plant tissue and differentiated plants which contain the regulatory region in this invention.

FIELD OF THE INVENTION

The present invention is related to isolated DNA sequences which act asregulatory regions in eukaryotic cells. More specifically, the presentinvention is related to isolated DNA sequences from maize which act asmale tissue-preferred regulatory regions and play a role in theexpression of genes in male tissues. The present invention is alsodirected to a method for conferring on a gene, which may or may not benormally expressed in male tissues, the ability to be expressed in amale tissue-preferred manner.

BACKGROUND OF THE INVENTION

Tissue- and temporal-specific gene expression and regulation are found,inter alia, during sexual reproduction in eukaryotes. In plantgametogenesis, important cytological and biochemical changes occurduring pollen development when the asymmetric mitotic division of thehaploid microspore results in the formation of two cells; each withdifferent developmental fates. The vegetative cell supports pollengrowth while the generative cell undergoes mitosis and develops intosperm cells. Messenger RNAs specific to both pathways within pollen havebeen identified in plants such as maize, tomato, tobacco, rice andpansy; and messages specific to pollen or to one or more other celltypes within anther such as tapetum, epidermis and stomium have alsobeen identified.

Pollen gene expression during differentiation involves an estimated24,000 genes (Willing, et al., "An Analysis of the Quantity andDiversity of mRNA's From Pollen and Shoots of Zea mays"; Theor. Appl.Genet.; Vol. 75; pp. 751-753; (1988)), however only 10% of clones from acDNA library are male-specific (Stinson, et al., "Genes Expressed in theMale Gametophyte and Their Isolation"; Plant Physiol.; Vol. 83; pp.442-447; (1987)) and the percentage of genes expressed in pollen thatare pollen-specific is between 5% and 80% (willing, et al., "An Analysisof the Quantity and Diversity of mRNA's From Pollen and Shoots of Zeamays"; Theor. Appl. Genet.; Vol. 75; pp. 751-753; (1988)). This complexprocess of microsporogenesis involves the sequential production of manygene products.

To date male-specific genes have been cloned from plants: two of these,the maize Ms45 gene (U.S. Pat. No. 5,478,369) and the Arabidopsis Ms2gene (Mark, G. M., et al., Nature: Vol. 363; pp. 715-717; (1993)), havebeen shown to be required for pollen development. Other examples ofmale-specific promoters in plants include ZM13, PG, and SGB6.

The Zm13 promoter is disclosed in U.S. Pat. No. 5,086,169. It consistsof 1315 base pairs and is from a pollen specific gene described byHanson, et al., Plant Cell; Vol. 1; pp. 173-179; (1989). This genehybridizes to mRNA found only in pollen.

Another pollen-specific promoter has been isolated and characterizedupstream of the pollen-specific polygalacturonase gene (PG) U.S. Pat. No5,412,085. This promoter region encompasses 2687 base pairs and isexpressed predominantly in pollen and emergent tassel, but not inpre-emergent tassel. U.S. Pat. No. 5,545,546, also from Allen, describesanother pollen-specific promoter from the maize polygalacturonase gene.It is only expressed in pollen and in emergent tassel.

U.S. Pat. No. 5,470,359 describes a regulatory region from the SGB6 geneof maize which confers tapetum specificity. The tissue of expression,the tapetum, is a layer of cells that surrounds the microsporogenouscells in the anther and provides nutrients thereto.

Nine anther-specific cDNA and genomic clones from tobacco are describedin U.S. Pat. No. 5,477,002. The cDNA clones were anther-specific byNorthern analysis, yet differed in developmental profiles. Clone Ant32is tapetal-specific.

European Pat. No. 0 420 819 Al describes the method of producing malesterile plants with the wun1 gene.

PCT WO 90/08825 describes anther-specific cDNAs TA13, TA26 and TA29 andtheir use in a male sterility system.

PCT WO 90/08825 explains male-sterility genes pMS10, pMS14 and pMS18 andtheir use with the GUS reporter gene.

U.S. Pat. No. 5,589,610 details a promoter corresponding toanther-specific cDNA and anther-preferred cDNA AC444.

The use of a plant promoter and an exogenous gene to effect a change inthe genetic make-up of plants is known in the art (U.S. Pat. Nos.5,432,068, 5,412,085, 5,545,546, 5,470,359 and 5,478,369) These patentsdiscuss plant expression cassettes with a tissue-specific promoterlinked to a gene to effect male sterility, fertility or otherwiseexpress a gene in a specific tissue. However, these patents do not teachthe use of this male tissue-preferred regulatory region or the use ofthis male tissue-preferred regulatory region with an exogenous gene as amethod of controlling male sterility.

The present invention is directed to a male tissue specific regulatoryregion and methods of using the same. Expression of an exogenous gene ina male tissue-preferred manner can mediate male fertility and is usefulin many systems such as in hybrid seed production.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forexpressing exogenous genes in a male tissue-preferred manner using anexpression vector that confers male tissue-preferred expression to anexogenous gene. This process may be used to restore (as in a malesterility system) or to otherwise impact fertility, as in hybrid seedproduction. It is a further object of this invention to provide a DNAregulatory region that confers male tissue-preferred gene expression. Itis also an object of this invention to provide a male tissue-preferredregulatory region or those with sequence identity thereto preferably ofabout 70%, 75%, or 80%, more preferably of about 85%, or 90%, and mostpreferably of about 95% or 99%.

It is an object of this invention to provide an isolated nucleic acidsequence encoding the Ms45 male tissue-preferred regulatory regions.

It is an object of this invention to provide an isolated nucleic acidsequence encoding an Ms45 male tissue-preferred regulatory region fromZea mays comprising a nucleic acid sequence shown in SEQ ID NO: 1 orthose with sequence identity thereto. It is also an object of thisinvention to provide an isolated nucleic acid sequence encoding a Ms45male tissue-preferred regulatory region from Zea mays comprising anucleic acid sequence shown in SEQ ID NO: 2 or those with sequenceidentity thereto.

It is an object of this invention to provide a recombinant expressionvector comprising the isolated nucleic acid sequence shown in SEQ ID NO:1, or those with sequence identity thereto, operably linked to anucleotide sequence encoding an exogenous gene such that said nucleotidesequence is expressed in a male tissue-preferred manner in such a waythat it promotes the expression of the exogenous gene.

It is an object of this invention to provide an exogenous gene, whereinsaid exogenous gene is Ms45.

It is an object of this invention to provide a method of producing atransformed plant that expresses an exogenous nucleotide sequence in amale tissue-preferred manner comprising the steps of introducing into aplant said exogenous nucleotide sequence operably linked to a maletissue-preferred regulatory region comprising a nucleotide sequencewhich is shown at SEQ ID NO: 1 or those with sequence identity thereto.The method wherein said introduction step may be performed bymicroprojectile bombardment, may utilize Agrobacterium or a transfervector comprising a Ti plasmid. Also, there may be more than one copy ofsaid exogenous nucleotide sequence operably linked to a maletissue-preferred regulatory region.

It is an object of this invention to provide a method wherein saidregulatory region expresses in a male tissue-preferred manner in tissuesselected from the group consisting of pollen, tapetum, anther, tassel,pollen mother cells and microspores.

It is an object of this invention to provide a transformed plantexpressing an exogenous nucleotide sequence in a male tissue-preferredmanner having an exogenous nucleotide sequence operably linked to a maletissue-preferred regulatory region shown at SEQ ID NO: 1 or those withsequence identity thereto. Said plant is a monocot or a dicot. Any plantcapable of being transformed may be used, including, for example, maize,sunflower, soybean, wheat, canola, rice and sorghum. This invention alsoprovides the transformed tissue of the transformed plant. By way ofexample, the tissue may be pollen, ears, ovules, anthers, tassels,stamens pistils and plant cells. The transformed plant may contain morethan one copy of said exogenous nucleotide sequence operably linked to amale tissue-preferred regulatory region.

It is an object of this invention to provide a method of mediatingfertility in a plant wherein the male tissue-preferred regulatory regionexpresses said exogenous nucleotide sequence such that fertility isimpacted. This exogenous nucleotide sequence can be any sequenceimpacting male fertility and can be, by way of example, a complementarynucleotidic unit encoding such antisense molecules as callase antisenseRNA, barnase antisense RNA and chalcone synthase antisense RNA, Ms45antisense RNA, or ribozymes and external guide sequences, or aptamers orsingle stranded nucleotides. The exogenous nucleotide sequence can alsoencode auxins, rol B, cytotoxins, diptheria toxin, DAM methylase,avidin, or may be selected from a prokaryotic regulatory system. Also,this exogenous nucleotide sequence is a male sterility gene or the Ms45structural gene and this plant is a monocot or a dicot.

It is an object of this invention to provide a method of producinghybrid seed, comprising planting in cross pollinating juxtaposition, amale fertile plant and a male infertile plant produced according to themethod above, allowing said cross pollination to occur and harvestingthe resulting seed. The plants can be maize plants.

These and other objects are achieved, in accordance with one embodimentof the present invention by the provision of an isolated DNA moleculewherein the DNA molecule comprises a nucleotide sequence shown at SEQ IDNO: 1.

In accordance with a further embodiment of the present invention, therehas been provided an expression vector comprising an exogenous gene,wherein the expression of the exogenous gene is under the control of amale tissue-preferred regulatory region, and where the product of theexogenous gene impacts male fertility.

In accordance with a further embodiment of the present invention, therehas been provided a method of using such an expression vector to producea male-sterile plant, comprising the step of introducing an expressionvector into plant cells, wherein the exogenous gene of the expressionvector may be a complementary nucleotidic unit such as antisensemolecules (callase antisense RNA, barnase antisense RNA and chalconesynthase antisense RNA, Ms45 antisense RNA), ribozymes and externalguide sequences, an aptamer or single stranded nucleotides. Theexogenous nucleotide sequence can also encode auxins, rol B, cytotoxins,diptheria toxin, DAM methylase, avidin, or may be selected from aprokaryotic regulatory system.

In accordance with a further embodiment of the present invention, therehas been provided a method of using a male tissue-preferred regulatoryregion to produce a male-fertile hybrid plant comprising the steps of:

a) producing a first parent male-sterile plant comprising an expressionvector that comprises a male tissue-preferred regulatory region and afirst exogenous gene, wherein the male tissue-preferred regulatoryregion controls the expression of the first exogenous gene, and whereinthe product of the first exogenous gene disrupts male fertility.

b) producing a second parent plant comprising an expression vector thatcomprises a second exogenous gene, wherein the regulatory regioncontrols the expression of the second exogenous gene so that it can beexpressed in male tissues;

c) cross-fertilizing the first parent with the second parent to producea hybrid plant, wherein the male tissues of the hybrid plant express thesecond erogenous gene, and wherein the product of the second exogenousgene prevents the disruption of the tassel function by the product ofthe first exogenous gene, thereby producing a male-fertile hybrid plant.

In accordance with a further embodiment of the present invention, therehas been provided a method of using a male tissue-preferred regulatoryregion to produce a male-fertile hybrid plant comprising the steps of:

a) producing a first parent male-sterile plant wherein a first geneinvolved in expression of male fertility is disrupted;

b) producing a second parent plant comprising an expression vector thatcomprises a male tissue-preferred regulatory region and an exogenousgene wherein the male tissue-preferred regulatory region controls theexpression of the exogenous gene so that it can be expressed in maletissues and could functionally complement the function of the genedisrupted in a);

c) cross-fertilizing the first parent with the second parent to producea hybrid plant, wherein the male tissues of the hybrid plant express theexogenous gene, and wherein the product of the exogenous gene preventsthe disruption of the tassel function, thereby producing a male-fertilehybrid plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of Ac 4.1 Ms45 genomic clone and restriction sites.

FIG. 2 is a plasmid map of PHP6045.

FIG. 3 is an autoradiogram of the primer extension products indicatingthe start of transcription of Ms45. Lanes labeled G, A, T, C, correspondto sequencing reactions with dideoxynucleotides ddGTP, ddATP, ddTTP, andddCTP, respectively. Lanes 1-4 correspond to primer extension reactionswith mRNA from (1) tassels, (2) leaves, (3) anthers, and (4) leaves.

FIG. 4 is a bar graph illustrating the stage specificity of the Ms45Male Tissue-Preferred Regulatory Region.

FIG. 5 illustrates tissue specificity illustrated by lack of activity innon-male tissue.

FIG. 6 shows an anther mRNA Northern analysis gel hybridized with themale fertility gene Ms45.

FIG. 7 shows the results of a mutational analysis of TATA box.

DISCLOSURE OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

1. Definitions

Sequence identity or similarity, as known in the art, are relationshipsbetween two polypeptide sequences or two polynucleotide sequences, asdetermined by comparing the sequences. In the art, identity also meansthe degree of sequence relatedness between two polypeptide or twopolynucleotide sequences as determined by the match between two stringsof such sequences. Both identity and similarity can be readilycalculated (Computational Molecular Biology; Lesk, A. M. ed.; OxfordUniversity Press, New York; (1988); Biocomputing: Informatics and GenomeProjects; Smith, D. W. ed.; Academic Press, New York; (1993); ComputerAnalysis of Sequence Data (Part I); Griffin, A. M. and H. G. Griffineds.; Humana Press, New Jersey; (1994); von Heinje, G., SequenceAnalysis in Molecular Biology; Academic Press; (1987); and SequenceAnalysis Primer; Gribskov, M. and J. Devereux eds.; M Stockton Press,New York; (1991)). While there exist a number of methods to measureidentity and similarity between two polynucleotide or two polypeptidesequences, both terms are well known to skilled artisans (von Heinje,G., Sequence Analysis in Molecular Biology; Academic Press; (1987);Sequence Analysis Primer; Gribskov, M. and J. Devereux eds.; M StocktonPress, New York; (1991); and Carillo, H., and D. Lipman, SIAM, J.Applied Math.; Vol. 48; pp. 1073; (1988)). Methods commonly employed todetermine identity or similarity between two sequences include, but arenot limited to those disclosed in Carillo, H., and D. Lipman, SIAM J.Applied Math.; Vol. 48; pp. 1073; (1988). Preferred methods to determineidentity are designed to give the largest match between the twosequences tested. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux J., et al., Nucleic AcidsResearch; Vol. 12(1); pp. 387; (1984)), BLASTP, BLASTN, and FASTA(Atschul, S. F., et al., J. Molec. Biol.; Vol. 215; pp. 403; (1990)).

Male tissue consists of tissues made of collections of cells that aredirectly involved or supportive of the reproduction of the male gametessuch as pollen, tapetum, anther, tassel, pollen mother cells andmicrospores. The tapetum is the tissue in the anther in closest contactwith the pollen mother cells and microspores and is likely involved withthe nutrition of the developing pollen grains. The Pollen mother cellsundergo two meiotic divisions that produce a tetrad of haploidmicrospores. Microspores undergo maturation into a pollen grain. Pollenor pollen grains are mature male gametophytes that can have the abilityto fertilize plants that are compatible. The anther is that portion ofthe stamen in which pollen is produced, the remainder of the stamenconsisting of the filament, from which the anther depends. The stamen isthe male organ of the flower.

The male tissue-preferred regulatory region is a nucleotide sequencethat directs a higher level of transcription of an associated gene inmale tissues than in some or all other tissues of a plant. For example,the Ms45 gene, described herein, is detected in anthers during quartet,quartet release and early uninucleate stages of development. For detailsregarding stages of anther development see Chang, M. T. and M. G.Neuffer, "Maize Microsporogenesis"; Genome; Vol. 32; pp. 232-244;(1989). The male tissue-preferred regulatory region of the Ms45 genedirects the expression of an operably linked gene in male tissues. Thepreferred tissues of expression are male, not for example, root orcoleoptile tissue. Predominant expression is in male tissues such as,but not limited to, pollen, tapetum, anther, tassel, pollen mother cellsand microspores. This male tissue-preferred expression refers to higherlevels of expression in male tissues, but not necessarily to theexclusion of other tissues.

To mediate is to influence in a positive or negative way or to influencethe outcome, such as with fertility or any other trait.

Male fertility is impacted when non-normal fertility is experienced;this can be as reduced fertility or increased fertility or fertilitythat is different in terms of timing or other characteristics.

Isolated means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living organismin its natural state is not "isolated," but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis "isolated", as the term is employed herein. For example, with respectto polynucleotides, the term isolated means that it is separated fromthe chromosome and cell in which it naturally occurs. As part of orfollowing isolation, such polynucleotides can be joined to otherpolynucleotides, such as DNAs, for mutagenesis, to form fusion proteins,and for propagation or expression in a host, for instance. The isolatedpolynucleotides, alone or joined to other polynucleotides such asvectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

An exogenous gene refers in the present description to a DNA sequencethat is introduced or reintroduced into an organism. For example, anygene, even the ms45 structural gene, is considered to be an exogenousgene, if the gene is introduced or reintroduced into the organism.

2. Isolation of a Male Tissue-Preferred Regulatory Region

Although anther-specific promoters and genes active in male tissues areknown in the art, (McCormick, et al., "Anther-Specific Genes: MolecularCharacterization and Promoter Analysis in Transgenic Plants," in PlantReproduction: From Floral Induction to Pollination; Lord, et al. eds.;pp. 128-135; (1989); Scott, et al., International ApplicationPublication No. WO 92/11379 (1992); van der Meer, et al., The PlantCell; Vol. 4; pp. 253; (1992)), there are no generally acceptedprinciples or structural criteria for recognizing DNA sequences thatconfer male tissue expression to gene expression in maize. Consequently,it is not possible to isolate a male tissue-preferred regulatory regiondirectly from a maize genomic library by screening for a consensussequence that confers male tissue-preferred expression.

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even highlystringent conditions (e.g., conditions represented by a wash stringencyof 35-40% Formamide with 5X Denhardt's solution, 0.5% SDS and 1x SSPE at37° C.; conditions represented by a wash stringency of 40-45% Formamidewith 5X Denhardt's solution, 0.5% SDS and 1X SSPE at 42° C.; andconditions represented by a wash stringency of 50% Formamide with 5XDenhardt's solution, 0.5% SDS and 1X SSPE at 42° C., respectively).Medium stringency in a standard hybridization of nucleic acids would beuseful in identifying the male tissue-preferred regulatory regionsdisclosed herein as well as other genes (see e.g. Sambrook, J., et al.,Molecular Cloning: a Laboratory Manual; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; (1982)). In general, sequences whichcode for a male tissue-preferred regulatory region will have sequenceidentity thereto of preferably 70%, 75%, or 80%, more preferably of 85%,or 90%, and most preferably of 95% or 99%.

Methods are readily available in the art for the hybridization ofnucleic acid sequences. Hybridization screening of plated DNA libraries(see e.g. Sambrook, J., et al., Molecular Cloning: a Laboratory Manual;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; (1982))or amplifying coding sequences using the polymerase chain reaction (seee.g. Innis, et al., PCR Protocols, a Guide to Methods and Applications;Academic Press; (1990)) are well known techniques for isolating genomicDNA.

Regulatory regions may be identified in the genomic subclones usingfunctional analysis, usually verified by the observation of reportergene expression in anther tissue and the reduction or absence ofreporter gene expression in non-anther tissue. This general approach isillustrated in Example 3, below. The possibility of the regulatoryregions residing "upstream" or 5' ward of the transcriptional start sitecan be tested by subcloning a DNA fragment that contains the upstreamregion and subcloning small fragments into expression vectors fortransient expression experiments. It is expected that smaller fragmentsmay contain regions essential for male-tissue preferred expression. Forexample, the essential regions of the CaMV 19S and 35S promoters havebeen identified in relatively small fragments derived from largergenomic pieces as described in U.S. Pat. No. 5,352,605.

In general, sequences which code for a male tissue-preferred regulatoryregion will have sequence identity thereto of preferably 70%, 75%, or80%, more preferably of 85%, or 90%, and most preferably of 95% or 99%.

Deletion analysis can occur from both the 5' and 3' ends of theregulatory region: fragments can be obtained by linker-scanningmutagenesis, mutagenesis using the polymerase chain reaction, and thelike (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)).The 3' deletions can delineate the male tissue-preferred regulatoryregion and identify the 3' end so that this essential region may then beoperably linked to a core promoter of choice. Once the essential regionis identified, transcription of an exogenous gene may be controlled bythe male tissue-preferred region of Ms45 plus a core promoter. The corepromoter can be any one of known core promoters such as a CauliflowerMosaic Virus 35S or 19S promoter (U.S. Pat. No. 5,352,605), Ubiquitin(U.S. Pat. No. 5,510,474), the IN2 core promoter (U.S. Pat. No.5,364,780), or a Figwort Mosaic Virus promoter (Gruber, et al., "Vectorsfor Plant Transformation" in Methods in Plant Molecular Biology andBiotechnology, Glick, et al. eds.; CRC Press; pp. 89-119; (1993)).Preferably, the promoter is the core promoter of a male tissue-preferredgene or the CaMV 35S core promoter. More preferably, the promoter is apromoter of a male tissue-preferred gene and in particular, the Ms45core promoter.

Further mutational analysis can introduce modifications of functionalitysuch as in the levels of expression, in the timing of expression or inthe tissue of expression. Mutations may also be silent and have noobservable effect.

3. Insertion of the region into an expression vector

The selection of an appropriate expression vector with which to test forfunctional expression will depend upon the host and the method ofintroducing the expression vector into the host and such methods arewell known to one skilled in the art. For eukaryotes, the regions in thevector include regions that control initiation of transcription andcontrol processing. These regions are operably linked to a reporter genesuch as the β-glucuronidase (GUS) gene or luciferase. Generaldescriptions and examples of plant expression vectors and reporter genescan be found in Gruber, et al., "Vectors for Plant Transformation" inMethods in Plant Molecular Biology and Biotechnology; Glick, et al. eds;CRC Press; pp. 89-119; (1993). Gus expression vectors and Gus genecassettes are commercially available from Clonetech, Palo Alto, Calif.,while luciferase expression vectors and luciferase gene cassettes areavailable from Promega Corporation, Madison, Wis. Ti plasmids and otherAgrobacterium vectors are described in Ishida, Y., et al., NatureBiotechnology; Vol. 14; pp. 745-750; (1996) and in U.S. Pat. No.5,591,616 Method for Transforming Monocotyledons, filed May 3^(rd),1994.

Expression vectors containing putative regulatory regions located ingenomic fragments can be introduced into intact tissues such as stagedanthers, embryos or into callus. Methods of DNA delivery includemicroprojectile bombardment, DNA injection, electroporation andAgrobacterium-mediated gene transfer (see Gruber, et al., "Vectors forPlant Transformation," in Methods in Plant Molecular Biology andBiotechnology, Glick, et al. eds.; CRC Press; (1993), U.S. Pat. No.5,591,616 Method for Transforming Monocotyledons, filed May 3^(rd),1994, and Ishida, Y., et al., Nature Biotechnology; Vol. 14; pp.745-750; (1996)). General methods of culturing plant tissues are foundin Gruber, et al., "Vectors for Plant Transfornmation," in Methods inPlant Molecular Biology and Biotechnology, Glick, et al. eds.; CRCPress; (1993).

For the transient assay system, staged, isolated anthers are immediatelyplaced onto tassel culture medium (Pareddy, D. R. and J. F. Petelino,Crop Sci. J.; Vol. 29; pp. 1564-1566; (1989)) solidified with 0.5%Phytagel (Sigma, St. Louis) or other solidifying media. The expressionvector DNA is introduced within 5 hours preferably bymicroprojectile-mediated delivery with 1.2μ particles at 1000-1100 Psi.After DNA delivery, the anthers are incubated at 26° C. upon the sametassel culture medium for 17 hours and analyzed by preparing a wholetissue homogenate and assaying for GUS or for lucifierase activity (seeGruber, et al., "Vectors for Plant Transformation," in Methods in PlantMolecular Biology and Biotechnology, Glick, et al. eds.; CRC Press;(1993)).

The above-described methods have been used to identify DNA sequencesthat regulate gene expression in a male tissue-preferred manner. Such aregion has been identified as the full length Ms45 male tissue-preferredregulatory region (SEQ ID No: 1). A TATA box mutation with sequenceidentity with the full length Ms45 male tissue-preferred regulatoryregion is identified in SEQ ID No: 2.

Thus, the present invention encompasses a DNA molecule having anucleotide sequence of SEQ ID No: 1 (or those with sequence identity)and having the function of a male tissue-preferred regulatory region.

A putative TATA box can be identified by primer extension analysis asdescribed in Example 2 below or in Current Protocols in MolecularBiology: Ausubel, F. M., et al., eds.; John Wiley and Sons, New York;pp. 4.8.1-4.8.5; (1987).

4. Use of a Male Tissue-Preferred Regulatory Region to Control Fertility

An object of the present invention is to provide a means to controlfertility using a male tissue-preferred regulatory region. Importantly,this male tissue-preferred regulatory region can control the expressionof an exogenous gene in anthers from quartet through early uninucleatestages of development. The practical significance of such timing is thatthe expression of a sterility-inducing gene during this developmentalstage will disrupt anther maturation early enough to permit visualverification of the function of the sterility-inducing system in thefield. It may also reduce the possibility that "breakers" (anthers thatshed pollen) will occur. Thus, the effects of the sterility-inducinggene would be evident in the production field at a sufficiently earlystage of development to allow either manual or mechanical detasseling ofany "fertile escapees" that result from a partial or total breakdown ofthe sterility-inducing system.

One approach to control male fertility is to manipulate gene expressionin the tapetum. The tapetum is a layer of cells that surroundsmicrosporogenous cells in the anther and likely provides nutrients, suchas reducing sugars, amino acids and lipids to the developing microspores(Reznickova, C. R., Acad. Bulg. Sci.; Vol. 31; pp. 1067; (1978); Nave,et al., J. Plant Physiol.; Vol. 125; pp. 451; (1986); Sawhney, et al.,J. Plant Physiol.; Vol. 125; pp. 467; (1986)). Ms45 is found to behighly expressed in the tapetal layer. Tapetal cells also produceβ(1,3)glucanase ("callase") which promotes microspore release (Mepham,et al., Protorlasma; Vol. 70; pp. 1; (1970)). Therefore, a delicaterelationship exists between the tapetum and the microsporogenous cells,and any disruption of tapetal function is likely to result indysfunctional pollen grains. In fact, lesions in tapetal biogenesis areknown to result in male sterility mutants (Kaul, "Male Sterility inHigher Plants" in Monographs on Theoretical and Applied Genetics;Frankel et al. eds.; Springer Verlag; Vol. 10; pp. 15-95; (1988)). Apremature or late appearance of callase during the development of thetapetum is also associated with certain types of male sterility (Warmke,et al., J. Hered.; Vol. 63; pp. 103; (1972)). Therefore, the callasegene can be used to disrupt male tissue function. Scott, et al., PCT WO93/02197 (1993), discloses the nucleotide sequence of a tapetum-specificcallase. Thus, a failure of the microspores to develop into maturepollen grains can be induced using a recombinant DNA molecule thatcomprises a gene capable of disrupting tapetal function under thecontrol of tapetum-specific regulatory sequences.

One general approach to impact male fertility is to construct anexpression vector in which the male tissue-preferred regulatory regionis operably linked to a nucleotide sequence that encodes a proteincapable of disrupting male tissue function, resulting in infertility.Proteins capable of disrupting male tissue function include proteinsthat inhibit the synthesis of macromolecules that are essential forcellular function, enzymes that degrade macromolecules that areessential for cellular function, proteins that alter the biosynthesis ormetabolism of plant hormones, structural proteins, inappropriatelyexpressed proteins and proteins that inhibit a specific function of maletissues.

For example, an expression vector can be constructed in which the maletissue-preferred regulatory region is operably linked to a nucleotidesequence that encodes an inhibitor of protein synthesis, which could bebut is not limited to a cytotoxin. Diphtheria toxin, for example, is awell-known inhibitor of protein synthesis in eukaryotes. DNA moleculesencoding the diphtheria toxin gene can be obtained from the AmericanType Culture Collection (Rockville, Md.), ATCC No. 39359 or ATCC No.67011 and see Fabijanski, et al., E.P. Appl. No. 90902754.2, "MolecularMethods of Hybrid Seed Production" for examples and methods of use. DAMmethylase, for example, is a well known enzyme from Escherichia coliwhich modifies the adenine residue in the sequence 5' GATC 3' to N⁶-methyl-adenine. Cigan and Albertsen describe how DAM methylase could beused to impact fertility in transgenic plants (PCT/US95/15229 Cigan, A.M. and Albertsen, M. C., "Reversible Nuclear Genetic System for MaleSterility in Transgenic Plants"). Another example of a protein whichdisrupts fertility is avidin as illustrated in U.S. pat. app. Ser. No.08/475,582 "Induction of Male Sterility in Plants by Expression of HighLevels of Avidin" by Howard, J. and Albertsen, M. C.

Alternatively, the disruption of tapetal function can be achieved usingDNA sequences that encode enzymes capable of degrading a biologicallyimportant macromolecule. For example, Mariani, et al., Nature: Vol. 347;pp. 737; (1990), have shown that expression in the tapetum of eitherAspergillus oryzae RNase-T1 or an RNase of Bacillus amyloliquefaciens,designated "barnase," induced destruction of the tapetal cells,resulting in male infertility. Quaas, et al., Eur. J. Biochem.; Vol.173; pp. 617; (1988), describe the chemical synthesis of the RNase-T1,while the nucleotide sequence of the barnase gene is disclosed inHartley, J. Molec. Biol.; Vol. 202; pp. 913; (1988).

RNase-T1 and barnase genes may be obtained, for example, by synthesizingthe genes with mutually priming long oligonucleotides. See, for example,Current Protocols in Molecular Biology; Ausubel, F. M., et al., eds.;John Wiley and Sons, New York; pp. 8.2.8 to 8.2.13; (1987). Also, seeWosnick, et al., Gene; Vol. 60; pp. 115; (1987). Moreover, currenttechniques using the polymerase chain reaction provide the ability tosynthesize very large genes (Adang, et al., Plant Molec. Biol.; Vol. 21;pp. 1131; (1993); Bambot, et al., PCR Methods and Applications; Vol. 2;pp. 266; (1993)).

In an alternative approach, pollen production is inhibited by alteringthe metabolism of plant hormones, such as auxins. For example, the rolBgene of Agrobacterium rhizogenes codes for an enzyme that interfereswith auxin metabolism by catalyzing the release of free indoles fromindoxyl-β-glucosides. Estruch, et al., EMBO J.; Vol. 11; pp. 3125;(1991) and Spena, et al., Theor. Appl. Genet.; Vol. 84; pp. 520; (1992),have shown that the anther-specific expression of the rolB gene intobacco resulted in plants having shriveled anthers in which pollenproduction was severely decreased. Therefore, the rolB gene is anexample of a gene that is useful for the control of pollen production.Slightom, et al., J. Biol. Chem.; Vol. 261; pp. 108; (1985), disclosethe nucleotide sequence of the rolB gene.

In order to express a protein that disrupts male tissue function, anexpression vector is constructed in which a DNA sequence encoding theprotein is operably linked to DNA sequences that regulate genetranscription in a male tissue-preferred manner. The generalrequirements of an expression vector are described above in the contextof a transient expression system. Here, however, the preferred mode isto introduce the expression vector into plant embryonic tissue in such amanner that an exogenous protein will be expressed at a later stage ofdevelopment in the male tissues of the adult plant. Mitotic stabilitycan be achieved using plant viral vectors that provide epichromosomalreplication.

An alternative and preferred method of obtaining mitotic stability isprovided by the integration of expression vector sequences into the hostchromosome. Such mitotic stability can be provided by themicroprojectile delivery of an expression vector to embryonic tissue(Gruber, et al., "Vectors for Plant Transformation," in Methods in PlantMolecular Biology and Biotechnology; Glick, et al. eds.; CRC Press;(1993)).

Transformation methodology can be found for many plants, including butnot limited to sunflower, soybean, wheat, canola, rice and sorghum(Knittel, N., et al., J. Plant Cell Rep.; Springer International,Berlin, W. Germany; Vol. 14(2/3); pp. 81-86; (1994); Chee, P. P., etal., Plant Physiol.; American Society of Plant Physiologists, Rockville,Md.; Vol. 91(3); pp. 1212-1218; (1989); Hadi, M. Z., et al., J. PlantCell Rep.; Springer International, Berlin, W. Germany; Vol. 15(7); pp.500-505; (1996); Perl, A., et al., Molecular and General Genetics; Vol.235(2-3); pp. 279-284; Zaghmout, O. M. F. and N. L. Trolinder, NucleicAcids Res.; IRL Press, Oxford; Vol. 21(4); pp. 1048; (1993); Chen, J. L.and W. D. Beversdorf, Theor. Appl. Genet.; Springer International,Berlin, W. Germany; Vol. 88(2); pp. 187-192; (1994); Sivamani, E., etal., Plant Cell Rep.; Springer International, Berlin, W. Germany; Vol.15(5); pp. 322-327; (1996); Hagio, T., et al., Plant Cell Rep.; Vol.10(5); pp. 260-264; (1991)) and are also known to those skilled in theart.

In order to select transformed cells, the expression vector contains aselectable marker gene, such as a herbicide resistance gene. Forexample, such genes may confer resistance to phosphinothricine,glyphosate, sulfonylureas, atrazine, imidazolinone or kanamycin.Although the expression vector can contain cDNA sequences encoding anexogenous protein under the control of a male tissue-preferredregulatory region, as well as the selectable marker gene under controlof constitutive promoter, the selectable marker gene can also bedelivered to host cells in a separate selection expression vector. Sucha "co-transformation" of embryonic tissue with a test expression vectorcontaining a male tissue-preferred regulatory region and a selectionexpression vector is illustrated below.

5. Induction of Sterility

In an alternative approach, male sterility can be induced by the use ofan expression vector in which the male tissue-preferred regulatoryregion is operably linked to a nucleotide sequence that encodes acomplementary nucleotidic unit. The binding of complementary nucleicacid molecules to a target molecule can be selected to be inhibitory.For example, if the target is an mRNA molecule, then binding of acomplementary nucleotide unit, in this case an RNA, results inhybridization and in arrest of translation (Paterson, et al., Proc.Nat'l. Acad. Sci.; Vol. 74; pp. 4370; (1987)). Thus, a suitableantisense RNA molecule, such as one complementary to Ms45 (U.S. Pat. No.5,478,369), would have a sequence that is complementary to that of anmRNA species encoding a protein that is necessary for male sterility(Fabijanski in "Antisense Gene Systems of Pollination Control For HybridSeed Production", U.S. pat. app. Ser. No. 08/288,734).

For example, the production of callase antisense RNA would inhibit theproduction of the callase enzyme which is essential for microsporerelease. In addition, male sterility can be induced by the inhibition offlavonoid biosynthesis using an expression vector that producesantisense RNA for the 3' untranslated region of chalcone synthase A gene(Van der Meer, et al., The Plant Cell, Vol. 4; pp. 253; (1992)). Thecloning and characterization of the chalcone synthase A gene isdisclosed by Koes, et al., Gene; Vol. 81; pp. 245; (1989), and by Koes,et al., Plant Molec. Biol.; Vol. 12; pp. 213; (1989).

Alternatively, an expression vector can be constructed in which the maletissue-preferred regulatory region is operably linked to a nucleotidesequence that encodes a ribozyme. Ribozymes can be designed to expressendonuclease activity that is directed to a certain target sequence inan mRNA molecule. For example, Steinecke, et al., EMBO J.; Vol. 11; pp.1525; (1992), achieved up to 100% inhibition of neomycinphosphotransferase gene expression by ribozymes in tobacco protoplasts.More recently, Perriman, et al., Antisense Research and Development;Vol. 3; pp. 253; (1993), inhibited chloramphenicol acetyl transferaseactivity in tobacco protoplasts using a vector that expressed a modifiedhammerhead ribozyme. In the context of the present invention,appropriate target RNA molecules for ribozymes include mRNA species thatencode proteins essential for male fertility, such as callase mRNA andMs45 mRNA.

In a further alternative approach, expression vectors can be constructedin which a male tissue-preferred regulatory region directs theproduction of RNA transcripts capable of promoting RNase P-mediatedcleavage of target mRNA molecules. According to this approach, anexternal guide sequence can be constructed for directing the endogenousribozyme, RNase P, to a particular species of intracellular mRNA, whichis subsequently cleaved by the cellular ribozyme (U.S. Pat. No.5,168,053; Yuan, et al., Science; Vol. 263; pp. 1269; (1994)).Preferably, the external guide sequence comprises a ten to fifteennucleotide sequence complementary to an mRNA species that encodes aprotein essential for male fertility, and a 3'-RCCA nucleotide sequence,wherein R is preferably a purine. The external guide sequencetranscripts bind to the targeted mRNA species by the formation of basepairs between the mRNA and the complementary external guide sequences,thus promoting cleavage of mRNA by RNase P at the nucleotide located atthe 5'-side of the base-paired region.

Another alternative approach is to utilize aptamer technology, where thecomplementary nucleotidic unit is a nucleotide that serves as a ligandto a specified target molecule (U.S. Pat. No. 5,472,841). This targetcould be a product essential for male fertility or a product disruptingmale fertility. Using this method, an aptamer could be selected for thetarget molecule, Ms45 or avidin for example, that would bind and inhibitexpression of the target. The nucleotide sequence encoding the aptamerwould be part of expression vectors constructed so that a maletissue-preferred regulatory region directs the production of theaptamer.

Sterility can also be induced by interruption of a gene important inmale fertility such as the Ms45 or the Ms2 gene (Mark, G. M., et al.,Nature; Vol. 363; pp. 715-717; (1993)). Methods of gene interruption arewell known in the art and include, but are not limited to, transposableelement insertion and mutation induction.

6. Restoration of Male Fertility in the Fl Hybrid

The above-described methods can be used to produce transgenicmale-sterile maize plants for the production of F1 hybrids inlarge-scale directed crosses between inbred lines. If the egg cells ofthe transgenic male-sterile plants do not all contain the exogenous genethat disrupts tapetal function, then a proportion of Fl hybrids willhave a male-fertile phenotype. On the other hand, F1 hybrids will have amale-sterile phenotype if the exogenous gene is present in all egg cellsof the transgenic male-sterile plants because sterility induced by theexogenous gene would be dominant. Thus, it is desirable to use a malefertility restoration system to provide for the production ofmale-fertile F1 hybrids. Such a fertility restoration system hasparticular value when the harvested product is seed or when crops areself-pollinating.

Also, such a fertility restoration system has particular value when themale tissue-preferred regulatory region is operatively linked to aninducible promoter such as in WO 89/10396 (Mariani, et al., Plants withModified Stamen Cells) and the inducible promoter is responsive toexternal controls. This linked male tissue-preferred regulatory regionconsists of a male tissue-preferred regulatory region, an induciblepromoter and an exogenous gene.

One approach to male fertility restoration would be to cross transgenicmale-sterile plants with transgenic male-fertile plants which contain afertility restoration gene under the control of a male tissue-preferredregulatory region. For example, Mariani et al, EP 0 344 029 crossedmale-fertile plants that expressed a barnase inhibitor, designated"barstar," with male-sterile plants that expressed barnase. Hartley, J.Mol. Biol.; Vol. 202; pp. 913; (1988), discloses the nucleotide sequenceof barstar.

Another approach would be to cross male-sterile plants containing adisruption in an essential male fertility gene, to transgenic malefertile plants containing the male tissue-preferred regulatory regionoperably linked to a non-disrupted copy of the fertility gene such asMs45 or Ms2 gene. The full sequence of the Ms45 gene is contained inU.S. Pat. No. 5,478,369 and Ms2 in Mark, G. M., et al., Nature; Vol.363; pp. 715-717; (1993).

Alternatively, male fertility restoration can be achieved by expressingcomplementary nucleotidic units such as toxin ribozymes or aptamers inmale-fertile plants to neutralize the effects of toxin in male-sterileplants. Thus, male fertility can be restored in the F1 hybrids byproducing a male-fertile transgenic plant that synthesizes a particularspecies of RNA molecule or polypeptide to counteract the effects of theparticular exogenous gene expressed in the male-sterile transgenicplants.

In an alternative method for restoring male fertility, transgenicmale-sterile plants contain an expression vector having a maletissue-preferred regulatory region, a prokaryotic regulatory region(from a prokaryotic regulatory system), and an exogenous gene that iscapable of disrupting tapetal function. Transgenic male-fertile plantsare produced that express a prokaryotic peptide under the control of amale tissue-preferred regulatory region. In the resulting F1 hybridsfrom the male-sterile and male-fertile cross, the prokaryotic peptidebinds to the prokaryotic regulatory sequence and represses theexpression of the exogenous gene which is capable of disrupting malefertility. An advantage of this method of fertility restoration is thatone form of transgenic male-fertile plant can be used to provide F1fertility regardless of the identity of the exogenous gene that was usedto disrupt tapetal function in the transgenic male-sterile plant.

For example, the LexA gene/LexA operator system can be used to regulategene expression pursuant to the present invention. See U.S. Pat. No.4,833,080 and Wang, et al., Mol. Cell. Biol.; Vol. 13; pp. 1805; (1993).More specifically, the expression vector of the male-sterile plant wouldcontain the LexA operator sequence, while the expression vector of themale-fertile plant would contain the coding sequences of the LexArepressor. In the F1 hybrid, the LexA repressor would bind to the LexAoperator sequence and inhibit transcription of the exogenous gene thatencodes a product capable of disrupting male fertility. These wouldinclude, but are not limited to, avidin, DAM methylase, diptheria toxin,RNase T, barnase, rol B and chalcone synthase A.

LexA operator DNA molecules can be obtained, for example, bysynthesizing DNA fragments that contain the well-known LexA operatorsequence. See, for example, U.S. Pat. No. 4,833,080 and Garriga, et al.,Mol. Gen. Genet.; Vol. 236; pp. 125; (1992). The LexA gene may beobtained by synthesizing a DNA molecule encoding the LexA repressor.Gene synthesis techniques are discussed above and LexA gene sequencesare described, for example, by Garriga, et al., Mol. Gen. Genet.; Vol.236; pp. 125; (1992). Alternatively, DNA molecules encoding the LexArepressor may be obtained from plasmid pRB500, American Type CultureCollection accession No. 67758. Those of skill in the art can readilydevise other male fertility restoration strategies using prokaryoticregulatory systems, such as the lac repressor/lac operon system or thetrp repressor/trp operon system.

7. Identification of Essential Parts of Regulatory Region

Identification of the essential parts of a regulatory region can beperformed by deleting, adding and/or substituting nucleotides in aregulatory region by methods well known to one skilled in the art. Suchvariants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis and mutagenesis using thepolymerase chain reaction (Directed Mutagenesis: A Practical Approach;IRL Press; (1991)).

A series of 5' deletions of a regulatory region can be constructed usingexisting restriction sites. The resulting promoter fragments can betested for activity using an expression vector as previously discussed.Further refinement and delineation may be obtained by making smallerchanges, preferably of about 50 or 30 nucleotides, more preferably ofabout 20 or 10 nucleotides and most preferably of about 5 or 1nucleotides, to the smallest restriction fragment that still confersproper expression upon the reporter construct (Directed Mutagenesis: APractical Approach; IRL Press; (1991)). These can be introduced into theexpression vector using introduced or natural restriction sites. Aseries of 3' deletions can also be generated as discussed above or byPCR or by methods well known to one skilled in the art (DirectedMutagenesis: A Practical Approach; IRL Press; (1991)). Furtherrefinement and delineation may be obtained by making smaller changes,preferably of about 50 or 30 nucleotides, more preferably of about 20 or10 nucleotides and most preferably of about 5 or 1 nucleotides, to thesmallest restriction fragment that still confers proper expression uponthe reporter construct (Directed Mutagenesis: A Practical Approach; IRLPress; (1991)).

These 5' and 3' deletions therefore will delineate the minimal regionessential for mimicking the proper tissue and temporal expression of thelonger regulatory region. In general, sequences which code for thisminimal region of a male tissue-preferred regulatory region will havesequence identity thereto preferably of about 70%, 75%, or 80%, morepreferably of about 85%, or 90%, and most preferably of about 95% or99%.

The following is presented by way of illustration and is not intended tolimit the scope of the invention.

EXAMPLE 1 Genomic Cloning and sequencing of Ms45 promoter

The Ac tagging and identification of the Ms45 cDNA and Northern analysisis described in U.S. Pat. No. 5,478,369.

A partial cDNA of Ms45 was used to screen a B73 maize genomic library.This library was made by cloning SAU3A1 partials into a BAMHI digestedgenomic cloning vector (Lambda Dash II, Stratagene, La Jolla, Calif.).Approximately 1×10⁶ plaques were screened using an E. coli strainsuitable for genomic DNA (ER1647, New England Biolabs, Mass.) as thehost. Clone AC4.1 was purified to homogeneity after three rounds ofscreening. Restriction mapping of AC4.1 showed the clone to be about 13kb in length and contained two internal BAMHI sites (FIG. 1). One ofthese sites was also found in the Ms45 partial cDNA. Two BAMHI fragmentswere subcloned to a cloning vector (Bluescript SK+, Stratagene, LaJolla, Calif.). The 5' end clone was about 3.5 kb in length andcorresponded to sequence upstream (5') of the internal BAMHI site. The3' end clone was 2.5 kb and contained Ms45 sequence downstream of theinternal BAMHI site. Concurrently, a putative full length Ms45 cDNA wasisolated and sequenced. By sequence comparison of the 5' end clone andthe Ms45 cDNA the putative translational start site was identified (FIG.1).

Sequencing of the Ms45 promoter region was accomplished using thedideoxy chain termination method of Sanger, F., et al., "DNA Sequencingwith Chain Terminating Inhibitors"; Proc. Nat'l. Acad. Sci.; Vol. 74;pp. 5463-5467; (1977). Genomic clone pac4.1-5' (FIG. 1) was sequencedusing the universal oligo and others that were sequence specific usingtechniques well known in the art.

The male tissue-preferred regulatory region had an NCOl site introducedat the start codon and was cloned as an NCOl fragment into apromoterless Luci expression vector. This new reporter vector wasdesignated as plasmid PHP6045 (FIG. 2) ATCC No: 97828 (Deposited Dec.12, 1996; American Type Culture Collection, 12301 Parklawn Dr.,Rockville, Md. 20852).

EXAMPLE 2 Primer Extension Analysis

Total RNA was isolated from maize tassels containing quartet throughearly uninucleate stage anthers. The total RNA was precipitated withethanol and MgCl₂. One milligram of total RNA was isolated and the polyA+ mRNA was purified by using oligo-dT cellulose. Poly A+ RNA was alsoisolated directly from 6 day old maize seedling leaves and maize anthersusing protocols known to those skilled in the art.

A sequencing ladder was prepared using a single stranded Ms45oligonucleotide and incorporation of 35S-dATP in a standard sequencingprocedure, using protocols well known to one skilled in the art.

Primer Extension was done according to the method below:

I. 5'-end labeling synthetic oligonucleotide primer.

    ______________________________________                                        Combined:  5 pmol primer N11916 (PHL11916) in 1.0 μl                          5 μl (50 μCi) gamma 32P-ATP                                             (>5000 Ci/mmole)                                                              0.7 μl 10X kinase buffer                                                   0.7 μl T4 polynucleotide kinase                                         incubated 37° C., 45 min                                                 Diluted with 20 μl TE and heated to 65° C. to inactivate          enzyme.                                                                            10X Kinase Buffer    To make 1 ml                                          0.5 M Tris-HCl, pH 7.6-8.0      0.5 ml of 1 M                                 5 mM spermidine                    0.05 ml of 0.1 M                           100 mMMgCl2                        0.1 ml of 1 M                              100 mM DTT                         0.5 ml of 0.5 M                            0.1 mg/ml gelatin or BSA           50 μl of 2 mg/ml                                                                      0.1 ml water                  ______________________________________                                    

II. Annealed primer and RNA

Kinased primers were annealed to mRNA from maize tassel, 6d maizeseedling leaves, maize anthers and 6d maize leaves. Mixed together onice were 2 μl mRNA, 1 μl kinased oligo, 2 μl 5X annealing buffer (1.25MKCl, 10 mM Tris, pH 7.9-8.15), and 1 μl 30 mM vanadyl. The total volumewas brought to 10 μl with 10 mM Tris, pH 8.15. This mixture was heatedto 65° C. and cooled to 55° C. for 4 hours period on thermocyclerheating block.

III. Primer extension

23 μl primer extension mix (see recipe below) and 0.4 μl reversetranscriptase (Superscript™, BRL, Md.) were added to each tube. This wasmixed by gently pipeting up and down and placed immediately in 48° C.and incubated 45 min. Primer Extension Mix consists of 10 mM MgCl2, 5mMDTT, 0.33 mM each dATP, dCTP, dGTP, dTTP and DEPC water.

300 μl ethanol was added and precipitated in -20 C. freezer overnight,then pelleted 30 minutes in a microfuge. Pellets were dried in a SpeedVac and dissolved in 6 μl of 0.1 NaOH/1 mM EDTA. Tube contents weremixed by pipetting and vortexing to insure that pellets were dissolved.These were left at room temp 2.5 hours, and 6 μl sequencing dye (Stopsolution from USB Sequencing kit) was added, and the solution wasdenatured at approximately 95° C. One half of the sample was loaded on6% denaturing polyacrylamide sequencing gel with stacking buffer and runat 55 Watts for 2 hours. The gel was dried in a gel dryer and exposed toKodak X-AR film. After a three day exposure, a transcription product wasobserved in the maize tassel mRNA primer extension reaction whichcorresponded to a deoxythymidine located 42 nucleotides upstream of thestart codon (FIG. 3). This position was designated as +1. A minor startof transcription was also identified at -3.

EXAMPLE 3 Determination of Stage and Tissue Specificity of the Ms45 MaleTissue-Preferred Regulatory Region

The full-length male tissue-preferred regulatory region (SEQ ID No: 1)was fused to the luciferase reporter gene from the firefly, Photinuspyralis, (DeWit, T. R., et al., Proc. Nat'l Acad. Sci. USA; Vol. 82; pp.7870-7873; (1985)) with the PinII-3' nontranslated region from potato(An, G., et al., "Functional Analysis of the 3' Control Region of thePotato Wound-Inducible Proteinase Inhibitor II Gene"; Plant Cell; Vol.1; pp. 115-122; (1989)). Maize anthers at various stages of developmentwere plated on tassel culture medium (Pareddy, et al., Theoret. Appl.Genet.; Vol. 77; pp. 521-526; (1989)), solidified with agar (Phytagar ®,Sigma, St. Louis). One of the three anthers from each floret was staged,and the remaining anthers were pooled by stage and plated formicroprojectile bombardment, typically eight anthers per plate. Theanthers were shot at 1100 p.s.i. with 1.8 p tungsten particles ontowhich was precipitated DNA of the Ms45 male tissue-preferred regulatoryregion-luciferase reporter construct. All anthers on a given plate wereat the same stage: premeiotic, meiosis I, meiosis II, quartet,microspore release, early uninucleate microspore, or mid-uninucleatemicrospore. Three repetitions were shot of each stage. Anthers wereincubated overnight at 26° C. for 18 hr. A crude extract was preparedwith the anthers from each plate and assayed for luciferase activity andprotein content. The luciferase activity, normalized to proteinconcentration, is graphed in FIG. 4 as a function of stage ofdevelopment. The major activity was at the quartet and microsporerelease stages of development, with minor activity in meiosis I and II,and barely detectable activity in the early uninucleate stage. Nosignificant activity above background was detected in premeiotic ormid-uninucleate anthers.

In addition, embryogenic callus, cultured on MS medium containing 2.0ug/ml of 2,4-D was bombarded in the same manner, except at 650 p.s.i.with particles coated with a luciferase reporter fused either to theMs45 male tissue-preferred regulatory region or to a maize ubiquitinpromoter (U.S. Pat. No. 5,510,474) and a uidA (GUS) reporter fused to amaize ubiquitin promoter. Luciferase was normalized to β-glucuronidase.As shown in FIG. 5, the Ms45 male tissue-preferred regulatory region wasincapable of driving transient expression in embryogenic callus andshoots, even though the ubiquitin promoter was expressed. Similarly,maize seeds, imbibed and germinated in distilled water for two days andplaced on wet filters, were subjected to microprojectile bombardment andtheir hypocotyls assayed for luciferase and β-glucuronidase. Theubiquitin regulatory region (promoter) was active, but the Ms45 maletissue-preferred regulatory region was not.

This result is paralleled by the results of RNA hybridization analysis.Maize anthers at various stages of development were collected andtreated as follows. One of the three anthers from each floret was fixedin (3:1 ethanol: glacial acetic acid) in a well of a microtiter plate,and two were frozen in liquid nitrogen in a well at the correspondingposition of another microtiter plate. Fixed anthers were staged; then,the corresponding frozen anthers were pooled by stage and polyA+ RNA wasisolated from 20 anthers (RNA Micro-Quick Prep kit, Pharmacia UppsilaSweden). Identical volumes of RNA from anthers at each pooled stage weresubjected to electrophoresis on 1.2% agarose in MOPSbuffer+formaldehyde. RNA samples were transferred by blotting to a nylonmembrane, fixed by UV cross-linking (Stratalinker, Stratagene Inc., LaJolla), and hybridized to a 32P-labeled probe fragment consisting of allof the Ms45 cDNA coding region and 3' region. The results shown in FIG.4 confirm steady state Ms45 transcript detectable in quartet throughearly uninucleate stages, and possibly as early as, but not earlierthan, telophase II in meiosis. Either transcript levels resulting fromMs45 male tissue-preferred regulatory region activity during meiosis donot accumulate sufficiently to be detected by RNA hybridization, or themeiotic stage male tissue-preferred regulatory region activity observedin transient assays does not occur in plants.

Thus the Ms45 male tissue-preferred regulatory region (SEQ ID NO: 1) wascharacterized as having male tissue-preferred expression from at leastquartet stage of anther development through quartet release, withlower-level expression possible in the meiotic and early uninucleatestages.

EXAMPLE 4 TATA Box Analysis

Within the 1388 bp fragment of DNA encoding the Ms45 maletissue-preferred regulatory region, the major start of transcription hasbeen identified at +1, a minor start of transcription has beenidentified at -3 relative to the major start of transcription, and aputative TATA box has been identified at -33 (CATTAAA). It was notedthat the sequence TAAAGAT at -30 could also be a candidate for theactual TATA box. This 1388 bp fragment was operably linked to a reportergene cassette comprising the luciferase coding region from firefly(Pareddy, et al., Theoret. Appl. Genet.; Vol. 77; pp. 521-526; (1989))followed by the 3'-nontranslated region from the proteinase inhibitor IIgene of potato. (An, G., et al., "Functional Analysis of the 3' ControlRegion of the Potato Wound-Inducible Proteinase Inhibitor II Gene";Plant Cell; Vol. 1; pp. 115-122; (1989)).

One way that is well known in the art to analyze TATA boxes is throughmutation. In another derivative, from one to six nucleotides of theputative TATA box were changed in a given derivative. A BGLII site wasintroduced at -38 altered the putative TATA box from CATTAAA to TATTAAA,which is a closer match to the canonical TATA box sequence TATATAA.

It will be appreciated by one skilled in the art that certainsubstitutions within the TATA box may affect the level of expression ofthe promoter without influencing tissue specificity. As shown in FIG. 7,the change in the TATA box associated with the BgIII site introduced at-38 dramatically increased transient expression levels in anthers andfurther suggests that the sequence at -33 is the authentic TATA boxIntroduction of a BGLII site at -40, -43, -51 or -53 did not increaseactivity of the promoter (data not shown), proving that the increaseobserved in the -38 BGLII site introduction was unrelated to the BGLIIsite per se.

Other modifications of the putative TATA box were introduced to furthertest for its functionality. Alteration of the putative TATA box sequencefrom CATTAAA to GATTAAA, CATGGAA or GGGCCCA all reduced the transientexpression level in anthers, further suggesting the importance of thissequence as a TATA box. Surprisingly, none of these mutations abolishedtransient activity; however, there have been reports of transientactivity in other systems in the absence of a TATA-like sequence andeven of TATA-less promoters (Guan, L., and J. G. Scandalios, Plant J.;Vol. 3; pp. 527-536; (1993); Close, P. S., "Cloning and MolecularCharacterization of Two Nuclear Genes for Zea mays MitochondrialChaperonin 60"; (Dissertation); Iowa State University, Ames, Iowa; pp.92, 128; (1993)).

While the foregoing describes preferred embodiments of the invention, itwill be understood by those skilled in the art that variations andmodifications may be made and still fall within the scope of theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1394 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CCATGGTGTC TCTATGAAAA AGATGAGTAC AATGTGTCTA TATCCGTTTT CT -            #TAGGGTCC     60                                                                 - - CTTCTTCTGC CTTATTACTG ACTGAATCGG GGTTACAAAA AACTTCCACG GG -            #TGCATGAT    120                                                                 - - CTCCATGTTC CACTTCTCCC ACCTCGCGTT GCACATTTCT TGGATGTCGG TG -            #GTTCCCAT    180                                                                 - - CTGACCGAGG CCCATCAGAC ACCTTTCGGG ACACCCATCA AGGGCCTTTC GG -            #ATGGCCCA    240                                                                 - - CGAGACGTAT CGGGTCGTGG TGATCCAGGG GATATATGTC CCCCACAATC GT -            #CACCTATA    300                                                                 - - TTATTATTCT TTAGATATTA TTTAATTTTT GGAAAAATAA CAAACTTATA CT -            #TTTGTGTA    360                                                                 - - GGGCCTCAGC ATAGATTTTC GCTTAGGGCC CAGAAATGCG AGGACCAGCC AT -            #GTCTAGTG    420                                                                 - - TCCACTATTG GCACTACCCA GAACAAGATT TAAAAAAATA ACCAAAGTAA CT -            #AATCCACT    480                                                                 - - CGAAAGCTAT CATGTAATGT TTAAAGAAAC ATCTATTAAA ACCACGATCC TC -            #TTAAAAAA    540                                                                 - - CAAGCATATT TCGAAAGAGA CAAATTATGT TACAGTTTAC AAACATCTAA GA -            #GCGACAAA    600                                                                 - - TTATATCGAA AGGTAAGCTA TGACGTTCAG ATTTTTCTTT TTCATTCTTG TT -            #ATTTTGTT    660                                                                 - - ATTGTTTTTA TATACATTTT CTTCTCTTAC AATAGAGTGA TTTTCTTCCG AT -            #TTTATAAA    720                                                                 - - ATGACTATAA AGTCATTTTT ATATAAGAGC ACGCATGTCG TAGATTCTCG TT -            #CAAAAATC    780                                                                 - - TTTCTGATTT TTTTAAGAGC TAGTTTGGCA ACCCTGTTTC TTTCAAAGAA TT -            #TTGATTTT    840                                                                 - - TTCAAAAAAA ATTAGTTTAT TTTCTCTTTA TAAAATAGAA AACACTTAGA AA -            #AATAGAGT    900                                                                 - - TGCCAGACTA GCCCTAGAAT GTTTTCCCAA TAAATTACAA TCACTGTGTA TA -            #ATTATTTG    960                                                                 - - GCCAGCCCCA TAAATTATTT AAACCGAAAC TGAAATCGAG CGAAACCAAA TC -            #TGAGCTAT   1020                                                                 - - TTCTCTAGAT TAGTAAAAAG GGAGAGAGAG AGGAAGAAAT CAGTTTTAAG TC -            #ATTGTCCC   1080                                                                 - - TGAGATGTGC GGTTTGGCAA CGATAGCCAC CGTAATCATA GCTCATAGGT GC -            #CTACGTCA   1140                                                                 - - GGTTCGGCAG CTCTCGTGTC ATCTCACATG GCATACTACA TGCTTGTTCA AC -            #CGTTCGTC   1200                                                                 - - TTGTTCCATC GTCCAAGCCT TGCCTATTCT GAACCAAGAG GATACCTACT CC -            #CAAACAAT   1260                                                                 - - CCATCTTACT CATGCAACTT CCATGCAAAC ACGCACATAT GTTTCCTGAA CC -            #AATCCATT   1320                                                                 - - AAAGATCACA ACAGCTAGCG TTCTCCCGCT AGCTTCCCTC TCTCCTCTGC CG -            #ATCTTTTT   1380                                                                 - - CGTCCACCAC CATG              - #                  - #                      - #   1394                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1394 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CCATGGTGTC TCTATGAAAA AGATGAGTAC AATGTGTCTA TATCCGTTTT CT -             #TAGGGTCC     60                                                                 - - CTTCTTCTGC CTTATTACTG ACTGAATCGG GGTTACAAAA AACTTCCACG GG -            #TGCATGAT    120                                                                 - - CTCCATGTTC CACTTCTCCC ACCTCGCGTT GCACATTTCT TGGATGTCGG TG -            #GTTCCCAT    180                                                                 - - CTGACCGAGG CCCATCAGAC ACCTTTCGGG ACACCCATCA AGGGCCTTTC GG -            #ATGGCCCA    240                                                                 - - CGAGACGTAT CGGGTCGTGG TGATCCAGGG GATATATGTC CCCCACAATC GT -            #CACCTATA    300                                                                 - - TTATTATTCT TTAGATATTA TTTAATTTTT GGAAAAATAA CAAACTTATA CT -            #TTTGTGTA    360                                                                 - - GGGCCTCAGC ATAGATTTTC GCTTAGGGCC CAGAAATGCG AGGACCAGCC AT -            #GTCTAGTG    420                                                                 - - TCCACTATTG GCACTACCCA GAACAAGATT TAAAAAAATA ACCAAAGTAA CT -            #AATCCACT    480                                                                 - - CGAAAGCTAT CATGTAATGT TTAAAGAAAC ATCTATTAAA ACCACGATCC TC -            #TTAAAAAA    540                                                                 - - CAAGCATATT TCGAAAGAGA CAAATTATGT TACAGTTTAC AAACATCTAA GA -            #GCGACAAA    600                                                                 - - TTATATCGAA AGGTAAGCTA TGACGTTCAG ATTTTTCTTT TTCATTCTTG TT -            #ATTTTGTT    660                                                                 - - ATTGTTTTTA TATACATTTT CTTCTCTTAC AATAGAGTGA TTTTCTTCCG AT -            #TTTATAAA    720                                                                 - - ATGACTATAA AGTCATTTTT ATATAAGAGC ACGCATGTCG TAGATTCTCG TT -            #CAAAAATC    780                                                                 - - TTTCTGATTT TTTTAAGAGC TAGTTTGGCA ACCCTGTTTC TTTCAAAGAA TT -            #TTGATTTT    840                                                                 - - TTCAAAAAAA ATTAGTTTAT TTTCTCTTTA TAAAATAGAA AACACTTAGA AA -            #AATAGAGT    900                                                                 - - TGCCAGACTA GCCCTAGAAT GTTTTCCCAA TAAATTACAA TCACTGTGTA TA -            #ATTATTTG    960                                                                 - - GCCAGCCCCA TAAATTATTT AAACCGAAAC TGAAATCGAG CGAAACCAAA TC -            #TGAGCTAT   1020                                                                 - - TTCTCTAGAT TAGTAAAAAG GGAGAGAGAG AGGAAGAAAT CAGTTTTAAG TC -            #ATTGTCCC   1080                                                                 - - TGAGATGTGC GGTTTGGCAA CGATAGCCAC CGTAATCATA GCTCATAGGT GC -            #CTACGTCA   1140                                                                 - - GGTTCGGCAG CTCTCGTGTC ATCTCACATG GCATACTACA TGCTTGTTCA AC -            #CGTTCGTC   1200                                                                 - - TTGTTCCATC GTCCAAGCCT TGCCTATTCT GAACCAAGAG GATACCTACT CC -            #CAAACAAT   1260                                                                 - - CCATCTTACT CATGCAACTT CCATGCAAAC ACGCACATAT GTTTCCTGAA CA -            #GATCTATT   1320                                                                 - - AAAGATCACA ACAGCTAGCG TTCTCCCGCT AGCTTCCCTC TCTCCTCTGC CG -            #ATCTTTTT   1380                                                                 - - CGTCCACCAC CATG              - #                  - #                      - #   1394                                                                __________________________________________________________________________

We claim:
 1. An isolated nucleotide acid encoding a regulatory regioncomprising a nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 2 orthose nucleotide sequences which hybridize to either of SEQ ID NO. 1 orNO. 2 under conditions of high stringency.
 2. A recombinant expressionvector comprising the isolated nucleic acid of claim 1, operably linkedto a nucleotide sequence encoding an exogenous gene such that saidexogenous gene is expressed in a male tissue-preferred manner.
 3. Amethod of producing a transformed plant that expresses an exogenousnucleotide sequence in a male tissue-preferred manner comprisingintroducing into a plant said exogenous nucleotide sequence operablylinked to a male tissue preferred regulatory region comprising thenucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 2 or those nucleotidesequences which hybridize to either of SEQ ID NO. 1 or NO. 2 underconditions of high stringency.
 4. The method according to claim 3wherein said introduction step is performed by microprojectilebombardment.
 5. The method according to claim 3 wherein saidintroduction step utilizes Agrobacterium.
 6. The method according toclaim 5 wherein said Agrobacterium comprises a Ti plasmid.
 7. The methodaccording to claim 3 wherein said regulatory region expresses in a maletissue-preferred manner in tissues selected from the group consisting ofpollen, tapetum, anther, tassel, pollen mother cells and microspores. 8.The method of claim 3 wherein a male-tissue preferred expressing portionof said male tissue-preferred regulatory region is present in more thanone copy.
 9. A method of controlling fertility in a plant comprisingproducing a transformed plant wherein the male tissue-preferredregulatory region of claim1, operably linked to an exogenous gene,expresses said exogenous nucleotide sequence such that fertility isimpacted.
 10. The method according to claim 3 wherein said exogenousnucleotide sequence comprises a DNA sequence encoding a prokaryoticregulatory system.
 11. The method according to claim3 wherein saidexogenous nucleotide sequence encodes a cytotoxic molecule.
 12. Themethod according to claim 3 wherein said exogenous nucleotide sequenceencodes avidin.
 13. The method according to claim 3 wherein saidexogenous nucleotide sequence encodes DAM methylase.
 14. The methodaccording to claim 3 wherein said exogenous nucleotide sequence encodessaid male tissue-preferred regulatory region operatively linked to acomplementary nucleotidic unit.
 15. The method according to claim 14wherein said complementary nucleotidic unit is selected from the groupconsisting of callase antisense RNA, barnase antisense RNA, chalconesynthase antisense RNA and Ms45 antisense RNA.
 16. The method accordingto claim 14 wherein said complementary nucleotidic unit is selected fromthe group consisting of ribozymes and external guide sequences.
 17. MThe method according to claim 9 wherein said exogenous nucleotidesequence encodes a product selected from the group consisting of auxins,rolB and diptheria toxin.
 18. The method according to claim 9 whereinsaid exogenous nucleotide sequence is a male sterility gene.
 19. Themethod according to claim 9 wherein the plant is a monocot.
 20. Themethod according to claim 9 wherein the plant is a dicot.
 21. A methodof producing hybrid seed, comprising planting in cross pollinatingjuxtaposition, a first male fertile plant and a second male infertileplant, the second male infertile plant produced by introducing into saidsecond plant an exogenous nucleotide sequence operably linked to a maletissue-preferred regulatory promoter comprising the nucleotide sequenceof SEQ ID NO. 1 or SEQ ID NO. 2 or those nucleotide sequences whichhybridize to either of SEQ ID NO. 1 or NO. 2 under conditions of highstringency such that said second plant is rendered male infertile,allowing said cross pollination to occur and harvesting the resultingseed.
 22. The method according to claim 21 wherein said plant is maize.23. A transformed plant expressing an exogenous nucleotide sequence in amale tissue-preferred manner comprising an exogenous nucleotide sequenceoperably linked to a regulatory region comprising a nucleotide sequenceof SEQ ID NO. 1 or SEQ ID NO. 2 or those nucleotide sequences whichhybridize to either of SEQ ID NO. 1 or NO. 2 under conditions of highstringency.
 24. A transformed plant of claim 23 wherein said exogenousnucleotide sequence is operably linked to a male tissue-preferredregulatory region, a male tissue-preferred expressing portion of whichmay be present in more than one copy.
 25. A transformed plant of claim23 wherein said plant is a monocot or a dicot.
 26. A transformed plantof claim 23 wherein said plant is selected from the group consisting ofmaize, sunflower, soybean, wheat, canola, rice and sorghum.
 27. Atransformed plant of claim 22 wherein said plant is selected from thegroup consisting of maize, sunflower, soybean, wheat, canola, rice andsorghum.
 28. Tissue of the transformed plant of claim 23 or claim 24.29. The transformed tissue of claim 28, wherein said tissue is selectedfrom the group consisting of pollen, ears, ovules, anthers, tassels,stamens pistils and plant cells.
 30. Transformed plant cells of thetransformed plant of claim 23.